Expression of the neuroendocrine peptide calcitonin (CT) and its receptor (CTR) is frequently elevated in prostate cancers (PCs), and activation of the CT-CTR axis in non-invasive PC cells induces an invasive phenotype. We aimed to link CT/CTR expression in prostate specimens to clinicopathological parameters of PC. We analyzed CT and CTR expression in cohorts of benign prostates and primary PCs with/without metastatic disease by immunohistochemistry. Furthermore, we correlated CT/CTR expression with several clinicopathological parameters. CT/CTR immunostaining in benign prostate acini was predominantly localized to basal epithelium. However, this spatial specificity was lost in malignant prostates. PC sections displayed a remarkable increase in cell populations expressing CT/CTR and their staining intensity. Tumors with higher CT/CTR expression consistently displayed metastatic disease and poor clinical outcome. High CT/CTR expression in primary prostate tumors may serve as a prognostic indicator of disease aggressiveness and poor clinical outcome.

The pharmacological inhibitors of poly(ADP-ribose) polymerase (PARP) family of proteins have shown promising results in preclinical studies and clinical trials as a monotherapy or in combination therapy for some cancers. Thus, usage of PARP-inhibitors (PARPi) in cancer therapy is bound to increase with time, but resistance of cancer cells to PARPi is also beginning to be observed. Here we review different known and potential mechanisms by which: (i) PARPi kill cancer cells; and (ii) cancer cells develop resistance to PARPi. Understanding the lethality caused by PARPi and the countermeasures deployed by cancers cells to survive PARPi will help us rationalize the use of this new class of drugs in cancer therapy.

Among the earliest responses of mammalian cells to DNA damage is catalytic activation of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Activated PARP-1 forms the polymers of ADP-ribose (pADPr or PAR) that posttranslationally modify its target proteins, such as PARP-1 and DNA repair-related proteins. Although this metabolism is known to be implicated in other repair pathways, here we show its role in the versatile nucleotide excision repair pathway (NER) that removes a variety of DNA damages including those induced by UV. We show that PARP inhibition or specific depletion of PARP-1 decreases the efficiency of removal of UV-induced DNA damage from human skin fibroblasts or mouse epidermis. Using NER-proficient and -deficient cells and in vitro PARP-1 assays, we show that damaged DNA-binding protein 2 (DDB2), a key lesion recognition protein of the global genomic subpathway of NER (GG-NER), associates with PARP-1 in the vicinity of UV-damaged chromatin, stimulates its catalytic activity, and is modified by pADPr. PARP inhibition abolishes UV-induced interaction of DDB2 with PARP-1 or xeroderma pigmentosum group C (XPC) and also decreases localization of XPC to UV-damaged DNA, which is a key step that leads to downstream events in GG-NER. Thus, PARP-1 collaborates with DDB2 to increase the efficiency of the lesion recognition step of GG-NER.

An accurate and sensitive detection of catalytic activation of poly(ADP-ribose) polymerase-1 (PARP-1) is required to be performed in a wide variety of samples because this activity plays a role in various cellular responses to DNA damage ranging from DNA repair to cell death, as well as in housekeeping functions, such as transcription. Since PARP-1 gene is expressed constitutively, its activation cannot be surmised from increased expression of its mRNA or protein, but by demonstrating the consequences of its catalytic -reaction which results in consumption of the substrate nicotinamide adenine dinucleotide (NAD(+)) and formation of three products, namely, polymer of ADP-ribose (pADPr or PAR), nicotinamide, and protons. Here, we describe various approaches commonly used in our laboratory for detection of PARP-1 activation in vivo (cells, tissues, and tumors), in situ, and in vitro via assessment of formation of pADPr, depletion of the substrate NAD, or formation of protons resulting in rapid and reversible intracellular acidification. It is important to note that although some other members of the PARP family can carry out the same catalytic reaction, many of these assays largely reflect PARP-1 activation in a vast majority of the experimental circumstances and more specifically in DNA damage responses. However, if required, PARP-1-specific action should be confirmed by use of PARP-1 knockout or RNAi-mediated knockdown approaches.

Common fragile sites (CFS) are specific chromosomal areas prone to form gaps and breaks when cells are exposed to stresses that affect DNA synthesis, such as exposure to aphidicolin (APC), an inhibitor of DNA polymerases. The APC-induced DNA damage is repaired primarily by homologous recombination (HR), and RAD51, one of the key players in HR, participates to CFS stability. Since another DNA repair pathway, the mismatch repair (MMR), is known to control HR, we examined the influence of both the MMR and HR DNA repair pathways on the extent of chromosomal damage and distribution of CFS provoked by APC and/or by RAD51 silencing in MMR-deficient and -proficient colon cancer cell lines (i.e., HCT-15 and HCT-15 transfected with hMSH6, or HCT-116 and HCT-116/3+6, in which a part of a chromosome 3 containing the wild-type hMLH1 allele was inserted). Here, we show that MMR-deficient cells are more sensitive to APC-induced chromosomal damage particularly at the CFS as compared to MMR-proficient cells, indicating an involvement of MMR in the control of CFS stability. The most expressed CFS is FRA16D in 16q23, an area containing the tumour suppressor gene WWOX often mutated in colon cancer. We also show that silencing of RAD51 provokes a higher number of breaks in MMR-proficient cells with respect to their MMR-deficient counterparts, likely as a consequence of the combined inhibitory effects of RAD51 silencing on HR and MMR-mediated suppression of HR. The RAD51 silencing causes a broader distribution of breaks at CFS than that observed with APC. Treatment with APC of RAD51-silenced cells further increases DNA breaks in MMR-proficient cells. The RNAi-mediated silencing of PARP-1 does not cause chromosomal breaks or affect the expression/distribution of CFS induced by APC. Our results indicate that MMR modulates colon cancer sensitivity to chromosomal breaks and CFS induced by APC and RAD51 silencing.

Prostate cancer is considered the most common cancer form among males in Western countries. Very limited options are available for the treatment of advanced metastatic prostate cancer. More than 50% of todays anticancer drugs are natural products or derived from a natural origin. To discover new entities with potential to treat prostate cancer at androgen-refractory stages, 36 structurally diverse natural products were screened using functional-based assays. The tested compounds were selected broadly from major secondary metabolites of plants, marine invertebrates, and fungi. These diverse entities were prescreened for their antiinvasive ability against prostate cancer cells, PC-3M, using spheroid disaggregation assay. Active representatives including three selected structural classes, a macrolide, a ?-carboline alkaloid, and a phenylmethylene hydantoin (PMH), were then tested for their ability to stabilize junctional complexes and enhance cell-cell adhesion of androgen independent prostate cancer cells. Transepithelial resistance (TER) and paracellular permeability assays were used to elicit the aforementioned properties. These studies led to the emergence of PMHs as a small molecule class from the marine sponge Hemimycale arabica with a unique potential to attenuate CT-stimulated prostate cancer growth, metastasis, paracellular permeability, and enhance TER and cell-cell adhesion of prostate cancer cells. The unique activities of PMHs were validated using several in vitro assays followed by in vivo testing in two mice models. A 3D QSAR was established using SYBYL 8.1-Comparative Molecular Field Analysis (CoMFA) model. This chapter includes the methodology for evaluation of structural and biological properties of new antiinvasive molecules with an exceptional potential to stabilize junctional complexes from diverse natural product sources.

Gene silencing by transient or stable RNA-interference (RNAi) is used for the study of apoptosis with an assumption that apoptotic events will not influence RNAi. However, we recently reported that stable RNAi, i.e., a permanent gene-knockdown mediated by shRNA-generating DNA vectors that are integrated in the genome, fails rapidly after induction of apoptosis due to caspase-3-mediated cleavage and inactivation of the endoribonuclease Dicer-1 that is required for conversion of shRNA to siRNA. Since apoptosis studies also increasingly employ transient RNAi models in which apoptosis is induced immediately after a gene is temporarily knocked down within a few days of transfection with RNAi-inducing agents, we examined the impact of apoptosis on various models of transient RNAi. We report here that unlike the stable RNAi, all forms of transient RNAi, whether Dicer-1-independent (by 21mer dsRNA) or Dicer-1-dependent (by 27mer dsRNA or shRNA-generating DNA vector), whether for an exogenous gene GFP or an endogenous gene poly(ADP-ribose) polymerase-1, do not fail for 2-3 days after onset of apoptosis. Our results reflect the differences in dynamics of achieving and maintaining RNAi during the early phase after transfection in the transient RNAi model and the late steady-state phase of gene-knockdown in stable RNAi model. Our results also sound a cautionary note that RNAi status should be frequently validated in the studies involving apoptosis and that while stable RNAi can be safely used for the study of early apoptotic events, transient RNAi is more suitable for the study of both early and late apoptotic events.

Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS) with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C(18)-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent.

Expression of calcitonin (CT) and its receptor (CTR) is elevated in advanced prostate cancer, and activated CT-CTR autocrine axis plays a pivotal role in tumorigenicity and metastatic potential of multiple prostate cancer cell lines. Recent studies suggest that CT promotes prostate cancer metastasis by reducing cell-cell adhesion through the disassembly of tight and adherens junctions and activation of beta-catenin signaling. We attempted to identify a class of molecules that enhances cell-cell adhesion of prostate cells and reverses the disruptive actions of CT on tight and adherens junctions. Screening several compounds led to the emergence of phenyl-methylene hydantoin (PMH) as a lead candidate that can augment cell-cell adhesion and abolish disruptive actions of CT on junctional complexes. PMH reduced invasiveness of PC-3M cells and abolished proinvasive actions of CT. Importantly, PMH did not display significant cytotoxicity on PC-3M cells at the tested doses. I.p. administered PMH and its S-ethyl derivative remarkably decreased orthotopic tumor growth and inhibited the formation of tumor micrometastases in distant organs of nude mice. PMH treatment also reduced the growth of spontaneous tumors in LPB-Tag mice to a significant extent without any obvious cytotoxic effects. By virtue of its ability to stabilize cell junctions, PMH could reverse the effect of CT on junctional disruption and metastasis, which strengthens the possibility of using PMH as a potential drug candidate for CT-positive androgen-independent prostate cancers.

Calcitonin, a neuroendocrine peptide, and its receptor are localized in the basal epithelium of benign prostate but in the secretory epithelium of malignant prostates. The abundance of calcitonin and calcitonin receptor mRNA displays positive correlation with the Gleason grade of primary prostate cancers. Moreover, calcitonin increases tumorigenicity and invasiveness of multiple prostate cancer cell lines by cyclic AMP-dependent protein kinase-mediated actions. These actions include increased secretion of matrix metalloproteinases and urokinase-type plasminogen activator and an increase in prostate cancer cell invasion. Activation of calcitonin-calcitonin receptor autocrine loop in prostate cancer cell lines led to the loss of cell-cell adhesion, destabilization of tight and adherens junctions, and internalization of key integral membrane proteins. In addition, the activation of calcitonin-calcitonin receptor axis induced epithelial-mesenchymal transition of prostate cancer cells as characterized by cadherin switch and the expression of the mesenchymal marker, vimentin. The activated calcitonin receptor phosphorylated glycogen synthase kinase-3, a key regulator of cytosolic beta-catenin degradation within the WNT signaling pathway. This resulted in the accumulation of intracellular beta-catenin, its translocation in the nucleus, and transactivation of beta-catenin-responsive genes. These results for the first time identify actions of calcitonin-calcitonin receptor axis on prostate cancer cells that lead to the destabilization of cell-cell junctions, epithelial-to-mesenchymal transition, and activation of WNT/beta-catenin signaling. The results also suggest that cyclic AMP-dependent protein kinase plays a key role in calcitonin receptor-induced destabilization of cell-cell junctions and activation of WNT-beta-catenin signaling.

The Red Sea sponge Hemimycale arabica afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin (1), (R)-5-(4-hydroxybenzyl)hydantoin (2), and (Z)-5-((6-bromo-1H-indol-3-yl)methylene)-hydantoin (3). The natural phenylmethylene hydantoin (PMH) 1 and the synthetic (Z)-5-(4-(ethylthio)benzylidene)-hydantoin (4) showed potent in vitro anti-growth and anti-invasive properties against PC-3M prostate cancer cells in MTT and spheroid disaggregation assays. PMHs 1 and 4 also showed significant anti-invasive activities in orthotopic xenograft and transgenic mice models. To study the effect of electronic and lipophilic parameters on the activity, a wide array of several substituted aldehydes possessing electron-withdrawing (+sigma), lipophilic (+pi), electron-donating (-sigma), and less lipophilic substituents (-pi) were used to synthesize several PMHs. Few des-phenylmethylenehydantoins and 2-thiohydanoins were also synthesized and the anti-invasive activities of all compounds were evaluated. Comparative molecular field analysis (CoMFA) was then used to study the 3D QSAR. Predictive 3D QSAR model with conventional r(2) and cross validated coefficient (q(2)) values up to 0.910 and 0.651 were established. In conclusion, PMH is a novel antimetastatic lead class with potential to control metastatic prostate cancer.

Mismatch repair (MMR) has been shown to control homologous recombination (HR) by aborting strand exchange between divergent sequences. We previously demonstrated that MMR-deficient tumour cells are more resistant to chromosomal damage induced by bleomycin (BLM) during the G(2) phase, likely due to the lack of the MMR inhibitory effect on HR. Aim of this study was to investigate whether inhibition of HR by the nucleoside analogue BVDU [(E)-5(2-bromovinyl)-2-deoxyuridine, brivudin], or silencing of genes involved in HR function, might affect sensitivity of MMR-deficient tumour cells to DNA damage induced by BLM in G(2). The results indicated that BVDU increased chromatid damage and DNA double strand breaks induced by BLM only in MMR-deficient MT-1, HL-60R, HCT116 cells, which are more resistant to BLM with respect to MMR-proficient TK-6, HL-60S and HCT116/3-6 lines. Silencing of RAD51, a key component of HR, increased sensitivity of MMR-deficient HCT-15 cells to BLM clastogenicity; in this case combined treatment with BVDU had no additional effect. Similarly, treatment with BVDU did not affect BLM clastogenicity in CAPAN-1 cells, characterized by a defective HR due to BRCA2 mutations. Conversely, BVDU increased chromatid breaks induced by BLM in HCT-15 cells transiently silenced for DNA-PK catalytic subunit, which plays a key role in non-homologous end joining. The BVDU-mediated increase of chromatid breaks in MMR-deficient cells did not depend on its previously reported inhibitory effect on poly(ADP-ribose) polymerase (PARP). In fact, it was observed also in cells stably silenced for PARP-1, which is responsible for most of cellular PARP activity. These data support the suggestion that the higher sensitivity of MMR-proficient versus MMR-deficient cells to BLM-induced chromatid breaks in the G(2) phase is a consequence of the inhibition of HR by MMR. In MMR-deficient cells, BVDU attenuates the repair of BLM-induced DSBs and this is likely to occur via inhibition of HR.

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.